Effect of a Nuclear Polyhedrosis

Virus of the Spruce Budwo~m on

the Metabolic Processes of Vertebrate Cells

by

Basi l M. Al'if and Peter Dobos

Forest Pest Management Institute

Sault See. Marie, Ontario

and

Department of Microbiology

University of Guelph

Guelph, Ontario

Report FPM-X-lS

Canadian Forestry Service

Environment Canada

August 1979

ISSN O)04-772X

ICi'~PDF

Copie 8 of t his report may be ob­tained from

DirectorForest Pest Management InstituteCanadian Forestry ServiceEnvironment CanadaBox 490, Sault Ste. Marie, OntarioP6A 5M?

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Developmen t ofTechniques to 0Effectiveness ASpecific. Pests

Determination 0

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Tec.hnologyTransfer

ABSTRACT

Mouse, fish and bird cell cultures were inoculated with a

nuclear polyhedrosis virus of the spruce budworm, Choristoneura

fumiferana. and at one. two or three days after inoculation radio-

active uridine, thymidine or leucine was added. At 2-h intervals

thereafter. the cells were processed and the synthesis of RNA, DNA

or protein was determined. The results, compared to those from

mock infected cells, were presented as qualitative incorporation of

the radioactive precursor into the respective macromolecule. The

data showed that the synthesis of RNA, DNA or protein increases

linearly with time and there was no difference between infected and

mock infected cells observed.

RESUME

L'auteur inocula des cellules de Vertebres (Souris. Poisson.

Oiseau) avec un virus de la polyh~drose nucleaire de la Tordeuse

des Bourgeons de l'Epinette, Choristoneura fumiferana. puis un. deux

ou trois jours apr~s l'inoculation. il ajouta de l'uridine, de la

-thymidine ou de la leucine radioactives. Ensuite. a intervales de

deux heures. les cellules furent travaillees et l'auteur effectua la

synthese de l'ARN. l'ADN ou la proteinp.. II presente les resultats.

compares a ceux de cellules pseudo-infectees. en tant qu'une 1n-

corporation qualitative du precurseur radioactif dans chaque macro-

molecule respective. II observe que la synthese de l'ARN, l'ADN ou la

proteine augmente lineairement avec Ie temps et que nulle difference

observable n'existe entre les cellules vraiment et faussement infectees.

TABLE OF CONTENTS

........ ..............................................

RESULTS AND DISCUSSION ••.

Radioactive Labelling.

.................... ........... ....................Page

1

2

2

2

2

4

4

8

12

................... .............. . ..... .... ...... .................... ...... .......... ......................... ......

................ ............... ...........

................................. .. . . . ... ..................................... ....................................... . .................

Cells ..

Vi rus ..

Protein synthesis ..

RNA synthesis •.

DNA synthesis.

MATERIALS AND METHODS.

INTRODUCTION

REFERENCES... . •••••• ••• .• .•••• • • . • • ••• ••• •••• ••• ••• •• ••• • • • • .••• 16

- 1 -

INTRODUCTION

The spruce budworm. Choristoneura fumiferana, is one of the

most serious forest pest in Eastern Canada and the North Eastern

United States. Every year this insect is responsible for the des­

truction of large areas of spruce-fir forest. So far, only chemical

insecticides have been used on a widescale basis to control in­

festations. However, the chemical insecticides currently used are

ecologically unacceptable since they affect many non-target organisms.

On the other hand, microbial insecticides generally affect only the

target insect and appear to be safe for vertebrates.

A nuclear polyhedrosis virus (NPV) of the spruce budworm has

been used experimentally as a biological insecticide and to date, over

1600 ha of infested forest have been sprayed with this virus. However,

before widespread use of this NPV as a registered forest control product

its effect on non-target organisms must be assessed.

In collaboration with the Ontario Veterinary College at the

University of Guelph safety tests were undertaken to ascertain the

pathogenicity of spruce budworm NPV to mammals and birds, (1.2). After

extensive testing the results proved conclusively that this insect

virus neither multIplies in vertebrates nor does it seem to alter

their metabolic processes. The effect of NPV on the metabolism of

vertebrates can be elucidated more definitively and accurately by in­

oculating cells grown in vitro with virus and studying macromolecular

synthesis in the presence of radioactive precursors. In this report

the effect of NPV on the qualitative biosynthesis of nucleic acids and

proteins is investigated.

- 2 -

MATERIALS AND METHODS

Virus

Virions were released from purified inclusion bodies by alkali

treatment and were then subjected to rate zonal and equilibrium

centrifugation on sucrose gradients (3). Each preparation was

bioassayed 1n fifth or sixth ius tar larvae and all virion preparations

were infectious.

Cells

The cells to be tested were L cells (mouse epithelial). chick

embryo fibroblasts (CEF) , RTG-2 (trout cells) and fat headed minnow

cells (FHM). They were grown on the bottom of clean. sterile glass

scintillation vials in complete minimum essential medium (HEM) at

the appropriate temperature until confluent monolayers were ob-

tained. One half of the cultures were infected with NPV (6-10 ~g

per culture flask) and the other half were mock infected. The virus

was allowed to adsorb for two hours. At one, two and three days after

infection 2 ml of medium, containing radioactive precursor, was added

to six infected and six uninfected cultures.

Radioactive Labeling

(a) In order to label intracellular proteins, leucine def­

3ficient medium was used to which H-leucine was added to contain 5~Ci/ml.

Leucine defficient medium consisted of 5% (v/v) of complete MEM (with-

out calf serum) and 95% leucine free ~ffiM. To this mixture 2% dialyzed

- 3 -

3calf serum was added followed by the addition of H-leucine.

(b) To label intracellula~ RNA, serum free ~ffiM containing

35~Ci/ml of H-uridine, was added to the cell cultures.

(c) For DNA labeling the cells were seeded at lower densities

to give semi-confluent monolayers in 1-2 days. This ensured active

DNA synthesis even after 3 days of incubation. The cells were

3labeled with H-thymidine (7~Ci/ml) in serum free MEM.

Infected and uninfected cultures were analyzed for acid pre-

cipitable radioactivity on days 1. 2 and 3 at 2-h intervals over

a period of 12 h. The radioactive medium was removed and the cells

were rinsed three times with cold phosphate buffered saline (PBS).

three times with ethanol and twice with ether. The precipitated

and fixed monolayers at the bottom of the vials were dried at room

temperature. 3 ml of scintillation cocktail was added and the amount

of incorporated radioactivity was assayed in a liquid scintillation

spectrometer.

- 4 -

RESULTS AND DISCUSSION

Protein Synthesis

L cells were inoculated with virion and separate cultures were

maintained at 28°C and 37°C. 3H-leucine was added at 1.2 and 3 days

after virus inoculation and the cells were examined for acid pre­

cipitable radioactivlty at 2-h intervals thereafter. Mock in­

fected cells were processed similarly (Fig. 1). The data presented

in this figure show that virus inoculated cells had the same rate

of protein synthesis as mock infected cells. Even) days after

infection the rate of protein biosynthesis remained the same. These

data show that NPV has no effect on mammalian cellular protein

synthesis.

The cells were maintained at 28°C because it 1s the optimum

temperature for NPV multiplication in the host insect. At 28°C the

level of protein synthesis is lo~er than that at 37°c which is ex­

pected since 28°c is considerably below the optimum temperature for

growth of mammalian cells (Fig. 1).

Simdlar data were obtained when using RTG-2 and FHM cells (Fig. 2).

RTG-2 cells cannot grow at temperatures much higher than 22°C and

FHM cells cannot grow at temperatures much lower than 28°c so tests

were conducted only at these temperatures.

Experiments were carried out for only 2 days at 32°C and 37°C

using CEF cells. since these cells begin to deteriorate after this

time (Fig. 3).

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- 8 -

It is interesting to note that the rate of protein biosynthesis in

CEF cells was unaltered when the cultures were maintained at 28°C or

37°C. The results summarized in Fig. 2 and 3 show that NPV had no

inhibitory or altering effect on avian or fish cell protein synthesis.

The above data demonstrate that these cells are refractory to

NPV as far as qualitative protein synthesis 1s concerned. Any effect

on the ribosomes. cellular mRNA or tRNA would have been manifested in

an altered rate of protein biosynthesis which was not observed.

RNA Synthesis

The experiments were similar to those used for monitoring the

rate of protein synthesis except that 3H-uridine. a specific RNA

metabolic precursor, was used. L cells grown at 37°C and 28°e

exhibited no difference in the rate of RNA synthesis between infected

and mock infected cultures (Fig. 4). It is clear, however. that the

rate of RNA synthesis at 28°e is much slower than that at 37°e since

the latter is the optimum temperature for vertebrate cells. Similar

data were obtained for CEF cells (Fig. 5) and for the two fish cell

lines (Fig. 6).

It is well documented that a number of viruses inhibit protein

and RNA synthesis in infected cultures (4). It is also clear that

NPV had no effect whatsoever on the quantitative RNA synthesis of

these four cell lines even at a temperature close to the optimum for

NPV multiplication.

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- 12 -

DNA Synthesis

The third parameter studied was the effect of NPV on the

synthesis of DNA in non-target cells. The experiments were carried

out using 3H-Thymidine. a DNA precursor, after inoculation with NPV.

The rate of formation of macromolecular DNA was monitored by the

rate of thymidine uptake. At days I, 2 and 3 after inoculation

3with NPV there was no difference in the rate of H-chymidine 10-

corporation between infected and uninfected cells (Fig. 7. 8 and 9).

It was also observed that at 28°e the rate of DNA synthesis in L

cells (Fig. 7) was much reduced compared to the rate observed at 37°C.

It must be emphasized here that reduced rate of DNA synthesis at 2a D e

is not due to virus infection since mock infected cells behaved

similarly at this temperature.

These cumulative data clearly demonstrated the inability of

spruce budworm NPV to alter or inhibit the rate of protein. RNA and

DNA synthesis in mammalian. avain or fish cell lines. These non-target

cells were maintained at their optimum temperatures as well as the

temperature optimum for NPV replication. NPV had no effect on the

cellular metabolic processes at either temperature. The conclusion

derived from these experiments is that NPV neither replicates nor is

partially expressed in non-target vertebrate cells.

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- 16 -

REFERENCES

1. Valli, V.E.O .• R. Irving and S. Beck. 1974. Mammalian toxicity

tests of the nuclear polyhedrosis virus of the spruce

budworm Choristoneura fumiferana. A research project

carried out by contract with the Dept. of Pathology,

Ontario Veterinary College, University of Guelph. Guelph,

Ontario.

2. Valli, V.E., C.M. Forsberg, J.e. Claxton and G.E. Fountain. 1975.

Avian toxicity tests of the nuclear polyhedrosis virus

of the spruce budworm Choristoneura fumiferana. A re­

search project carried out by contract with th~ Dept. of

Pathology. Ontario Veterinary College, University of

Guelph, Guelph. Ontario.

3. Arif, B.M. and K.W. Brown. 1975. Purification and properties

of a nuclear polyhedrosis virus from Choristoneura

fumiferana. Can. J. Microbial. 21: 1224-1231.

4. Davis, B.D., R. Dulbecco, R.N. Eisen. H.S. Ginsberg and W.B.

Wood. 1973. Multiplication and Genetics of Animal

Viruses. In "Microbiology" pp. 1140-1170. Harper aod

Row Publishers.